5-Fu对A549细胞NKG2D配体MICA/B启动子活性的作用

目的：克隆A549细胞NKG2D配体MICA/B的启动子,分析其活性,并研究5-Fu对MICA/B启动子活性的调节作用。方法：PCR方法扩增人MICA/B的启动子及其5个截短体基因,用双荧光报告基因系统进行启动子活性分析,并测定不同浓度5-Fu处理对启动子活性的影响。结果与结论：克隆了人MICA启动子（-455/＋4）、MICB启动子（-470/＋40）及其5个截短体基因,MICA的相对活性分别为：3.61,2.26,1.63,0.313,0.711,0.663;MICB的相对活性分别为：17.49,10.11,7.398,0.822,0.997,0.49。用20,40,80,160,320μg/ml的5-Fu处理A549细胞8h后,MICA启动子活性分别是未处理的1.69,1.48,1.62,1.55,1.78倍,MICB启动子活性分别是未处理的1.44,1.87,1.38,1.19,1.25倍。表明不同浓度5-Fu处理A549细胞后MICA/B启动子相对活性上升。     

贝伐单抗对耐药鼻咽癌细胞凋亡的影响

目的体外观察贝伐单抗（bevacizumab）对耐药鼻咽癌细胞凋亡的影响。方法以人鼻咽癌低分化细胞系CNE2及其顺铂（DDP）耐药细胞亚系CNE2/DDP为研究对象,在体外用不同浓度的贝伐单抗和DDP处理细胞,MTT法检测两种药物对CNE2/DDP的杀伤效应,流式细胞术测定CNE2和CNE2/DDP的凋亡率,半定量RT-PCR检测CNE2、CNE2/DDP及贝伐单抗处理的CNE2/DDP细胞中bcl-2、bax基因表达的变化。结果10μg/ml贝伐单抗对CNE2/DDP细胞的杀伤率（5.2%±4.3%）与空白对照组相似（0.0%±4.1%,P=0.180）,10μg/ml贝伐单抗分别与0.1、0.2μg/ml DDP联合的杀伤率均高于单用DDP（42.3%±6.5%vs34.4%±5.4%,P=0.041；62.6%±5.5%vs50.0%±5.9%,P=0.009）。10μg/ml贝伐单抗＋0.1μg/ml DDP联合作用后CNE2/DDP细胞的凋亡率（87.3%±3.4%）明显高于单用DDP（50.6%±8.8%,P〈0.05）。半定量RT-PCR检测显示CNE2、CNE2/DDP及贝伐单抗处理的CNE2/DDP中bcl-2的相对表达量分别为0.613、0.952和0.135,bax的相对表达量分别为0.665、0.387和1.751。结论贝伐单抗可以显著增加耐药细胞CNE2/DDP对DDP的药物敏感性；耐药细胞表现为凋亡抑制,而贝伐单抗能促进其凋亡。     

高、低表达三磷酸腺苷结合转运蛋白G超家族成员2的人鼻咽癌耐药细胞生物学特性差异研究

目的 探讨高表达与低表达三磷酸腺苷(ATP)结合转运蛋白G超家族成员2(ABCG2)的人多药耐药鼻咽癌CNE2/DDP细胞(简写作ABCG2High细胞ABCG2Low细胞)的生物学差异,分析两种细胞的耐药特点.方法 分离ABCG2High和ABCG2Low细胞,检测其体外克隆形成率并分析细胞周期.流式细胞术(FCM)检测两种细胞传代前及传至第5代时ABCG2阳性表达率的差异.RT-PCR检测耐药基因ABCG2、Bc1-2、MDR1、MRP、MGMT的mRNA表达差异.MTT法检测两种细胞在1～14d的吸光度,绘制生长曲线;检测两种细胞对氟尿嘧啶、顺铂、长春新碱、卡铂、表阿霉素、柔红霉素、紫杉醇、丝裂霉素等化疗药物耐药性的差异.结果 ABCG2High和ABCG2细胞在7d内的克隆形成率分别为25.24%±1.18%和16.28%±1.11%.FCM分析显示,ABCG2High的S期细胞占41.5%,ABCG2Low的G1M0期细胞占63.9%.FCM分离后,ABCG2的表达率在ABCG2High细胞为98%,在ABCG2Low细胞为2%;两种细胞传至第5代,ABCG2在前者的表达率降低为7s%,在后者升高至20%.RT-PCR显示,ABCG2 mRNA在ABCG2High细胞中的表达明显高于ABCG2low细胞,其他耐药基因在两种细胞中的表达无差异.生长曲线显示,ABCG2High细胞早期生长速度较ABCG2Low细胞快,后期生长速度逐渐减慢.耐药谱分析表明,ABCG2High细胞对8种抗肿瘤药物的IC50值高于ABCG2Low细胞两倍以上,两者差异有统计学意义(P＜0.05).结论 ABCG2High细胞是一种快周期细胞,而ABCG2Low细胞为慢周期细胞,前者更具有多药耐药的特点,可用于ABCG2介导的鼻咽癌细胞非经典多药耐药的研究.     

分化抑制因子1对鼻咽癌细胞增殖的影响及其途径

目的探讨分化抑制因子1(Id1)对鼻咽癌细胞增殖和凋亡的影响及其机制。方法体外培养人鼻咽癌细胞NP69、CNE1。合成Id1小分子干扰RNA(siRNA),构建Id1表达载体并分别转染鼻咽癌细胞NP69、CNE1。收集Id1siRNA及对照质粒Scramble转染72h的NP69细胞(分别命名为NP69si-Id1、NP69-NC),pcDNA3.1-Id1及对照pcDNA3.1转染48h的CNE1细胞(分别命名为CNE1-Id1、CNE1-Vector),采用RT-PCR及Western blotting检测Id1的表达情况。采用MTT法比较顺铂(DDP)对NP69si-Id1、NP69-NC和CNE1-Id1、CNE1-Vector增殖的影响。采用CalcuSyn软件计算各组细胞对DDP的IC50值。将1μg/ml DDP与鼻咽癌细胞共同孵育24h后,采用Western blo ing检测凋亡蛋白Caspase-3、Bax、Bad的表达及蛋白激酶B(Akt)Thr308和Ser473的磷酸化情况。结果与NP69-NC比较,NP69si-Id1的Id1 mRNA和蛋白水平明显降低;与CNE1-Vector比较,CNE1-Id1的Id1 mRNA及蛋白水平明显升高。NP69si-Id1细胞对DDP的IC50值(0.207±0.008μg/ml)明显低于NP69-NC(0.405±0.009μg/ml,P<0.05),CNE1-Id1细胞对DDP的IC50值(0.671±0.012μg/ml)明显高于CNE1-Vector(0.445±0.008/ml,P<0.05)。Western blotting检测结果显示,与NP69-NC比较,NP69si-Id1的Caspase-3断裂带增多,Akt Thr308和Ser473磷酸化减少;与CNE1-Vector比较,CNE1-Id1的Caspase-3断裂带减少,Akt Thr308和Ser473磷酸化增加。结论 Id1可促进鼻咽癌细胞的生长,抵抗DDP诱导的细胞凋亡,其机制可能与增加Akt磷酸化有关。     

肿瘤干细胞标志物ABCG2、CK19和P63在皮肤日光性角化病、Bowen病及鳞状细胞癌中的表达差异及意义

目的检测肿瘤干细胞标志物ABCG2、CK19和P63在皮肤鳞状细胞癌及其癌前病变组织中的表达及意义。方法选取2010年10月-2011年8月在中国医学科学院皮肤病研究所经病理学确诊的组织学标本37例,其中日光性角化病(AK)10例,Bowen’s病(BD)14例,鳞状细胞癌(SCC)13例。采用免疫组织化学MaxVision法检测ABCG2、CK19、P63在各类病变组织中的表达及程度,计算各分子在病变组织中的阳性表达百分率。结果 ABCG2和CK19的阳性反应物定位于细胞膜和细胞质,在SCC中两种分子的阳性细胞率(22.1%±12.6%,23.5%±18.5%)显著高于AK(7.1%±6.6%,5.7%±4.5%)和BD(10.8%±9.1%,6.6%±4.9%,P﹤0.01),而AK与BD比较差异无统计学意义(P>0.05)。AK、BD和SCC中ABCG2和CK19阳性病例的比例逐渐增加(ABCG2为40.0%、71.4%、84.6%,CK19为40.0%、50.0%、92.3%)。P63阳性反应物定位于细胞核,在AK、BD和SCC的所有病例中均呈阳性,其阳性细胞分别为50.5%±17.1%、56.3%±19.8%、43.5%±21.6%,三者比较差异无统计学意义(P>0.05)。结论 ABCG2、CK19和P63阳性表达程度可能与皮肤鳞状细胞癌的发生发展有关,可作为临床鳞癌患者预后评估的参考。     

ABCG2抑制剂联合光动力疗法对胶质瘤干细胞杀伤作用的研究

目的:观察ABCG2抑制剂联合光动力疗法对胶质瘤干细胞的作用。方法:将培养对数期的U251胶质瘤细胞系及其干细胞均分为激光组、光敏剂组、光动力组、光动力联合ABCG2抑制剂组以及单纯ABCG2抑制剂组,将未进行任何处理的细胞作为对照组。(1)观察不同处理组在相同时间和相同光照强度条件下U251细胞及其干细胞形态学变化。(2) MTT法检测各组细胞的生存率。(3) Western-blot检测各组处理前后细胞中ABCG2蛋白的表达情况。(4)TUNEL凋亡实验检测各组细胞的凋亡情况,比较加入ABCG2抑制剂后细胞凋亡情况的差异。结果:(1)U251及其干细胞在光动力组和光动力联合ABCG2组表现出明显的细胞回缩等改变,干细胞对光动力更加敏感。(2) PDT组和PDT+ABCG2组细胞生存率存在明显差异(P 0.05)。(3) PDT+ABGC2的干细胞处理组较单纯光动力组细胞凋亡更加明显。其余组未见明显差异。(4)干细胞中存在明显ABCG2蛋白的高表达,加入抑制剂后ABCG2表达明显降低,细胞凋亡更明显。结论:ABCG2抑制剂可以明显增加光动力对胶质瘤干细胞的杀伤作用。     

青蒿琥酯逆转Eca109/ABCG2细胞对阿霉素耐药的作用及机制

目的研究青蒿琥酯(Art)逆转食管癌耐药细胞Eca109/ABCG2对阿霉素(ADM)耐药的作用及其机制。方法采用不同浓度的青蒿琥酯(Art,0、0.01、0.1、1μmol/L),阿霉素(ADM,0、0.02、0.2、2、20mg/L),Art(0.01、0.1、1μmol/L)+ADM(0.02、0.2、2、20mg/L)处理Eca109/ABCG2细胞48h,然后以MTT法检测细胞生长抑制率。采用ADM(0、0.02、0.2、2mg/L),Art(0、0.01、0.1、1μmol/L)和ADM(0.2mg/L)+Art(0.01、0.1、1μmol/L)处理细胞,48h后以流式细胞术(FCM)检测细胞凋亡率及细胞内ADM含量,72h后检测细胞内ABCG2蛋白的表达。结果与单独应用Art或ADM相比,Art+ADM处理后,Eca109/ABCG2细胞的生长抑制率、凋亡率和细胞内ADM含量均显著增加(P<0.05),而细胞内ABCG2蛋白表达显著降低(P<0.05)。结论 Art可通过下调Eca109/ABCG2细胞ABCG2蛋白表达,增加细胞内ADM的药物浓度,从而增强疗效、逆转耐药。     

Apoptin与顺铂联用对膀胱癌T24细胞具有协同杀伤作用

目的探讨pApoptin-EGFP质粒转染与顺铂(DDP)联用对体外培养的膀胱癌T24细胞的协同杀伤作用。方法脂质体介导pApoptin-EGFP瞬时转染与梯度浓度DDP共同处理膀胱癌T24细胞,流式细胞仪检测细胞增殖变化;pApoptin-EGFP瞬时转染与10μg/ml的DDP处理T24细胞,Annexin V法检测凋亡变化;金氏法分析两种抗肿瘤方式的协同作用。结果 DDP、pEGFP-N1+DDP和pApoptin-EGFP+DDP 3组的IC50分别为10.61、7.9和2.4μg/ml,以金氏法计算,确认pApoptin-EGFP质粒转染与DDP处理对T24的抑制增殖效应具有协同作用,而转染空质粒与DDP干预仅仅为简单相加。同法检测DDP、pApoptin-EGFP、pApoptin-EGFP+DDP 3组的凋亡率分别为(6.96±1.32)%、(19.55±1.09)%和(32.5±1.15)%,3组凋亡率相互间均具有显著性差异(P<0.05)。按金氏法计算,确认转染pApoptin-EGFP重组质粒与10μg/ml浓度DDP处理对T24细胞具有协同的促凋亡作用。结论 Apoptin基因转染与顺铂联用对T24肿瘤抑制增殖及诱导凋亡具有协同作用。     

模拟失重对人自然杀伤细胞毒活性的影响

目的 研究模拟失重状态下人NK细胞的细胞毒活性并对其活性变化的机制进行初步的探讨.方法 采用高均一性的人NK细胞作为模型,以回转细胞培养模拟失重状态.人NK细胞经历48 h和72 h的模拟失重后,测定其杀伤率,并在mRNA水平检测NK细胞穿孔素基因、激活性受体基因以及抑制性受体基因的表达水平.结果 模拟失重48 h和72 h的人NK细胞,其杀伤率比对照组分别下降了16%(P＜0.05)和26%(P＜0.05).通过对模拟失重72 h NK细胞mRNA的定量分析,发现激活性受体NKG2D基因表达下调,抑制性受体NKG2A基因表达上调,而穿孔素基因表达没有显著性变化.经历72 h模拟失重的人NK细胞,回到正常条件下培养6 d后,其细胞毒活性才恢复至正常水平.结论 模拟失重可以导致人NK细胞杀伤活性的下降,其原因可能与NKG2A及NKG2D受体表达的变化有关.     

葡萄籽原花青素对卵巢癌裸鼠耐药移植瘤的抑制作用观察

目的探讨葡萄籽提取物原花青素(GSPE)对人卵巢癌顺铂耐药细胞COC1/DDP裸鼠皮下移植瘤的抑制作用及其机制。方法 30只COC1/DDP裸鼠建立皮下移植瘤模型,随机分为GSPE高浓度组、GSPE低浓度组、对照组(每组10只),分别以200mg/kgGSPE、100mg/kgGSPE及生理盐水灌胃[10ml/(kg·d)]。3周后处死裸鼠,测量各组移植瘤体积,流式细胞术(FCM)分析细胞凋亡率,RT-PCR、Western blotting检测肿瘤细胞Livin和caspase-3的mRNA、蛋白表达情况。结果 GSPE高、低浓度组皮下移植瘤体积(分别为351.23±15.52、432.51±18.36mm3)明显小于对照组(594.49±16.33mm3,P0.05)。FCM检测显示GSPE高、低浓度组肿瘤细胞凋亡率分别为66.25%、29.31%,明显高于对照组(1.03%,P0.01)。与对照组相比,GSPE能明显降低Livin的mRNA、蛋白表达水平(P0.01或P0.05),增加caspase-3的mRNA、蛋白表达水平(P0.01或P0.05)。结论 GSPE可能是通过降低Livin的表达、增强caspase-3的表达而显著抑制COC1/DDP细胞移植瘤的生长,促进COC1/DDP细胞的凋亡。     

Synthesis of no-carrier-added fluorine- 18 2-fluoro-2-deoxy-D-glucose

A new synthetic procedure for the preparation of fluorine- 18 2-fluoro-2-deoxy-glucose has been developed. This procedure offers the advantages of flexibility in the source of the fluorine- 18, high yields, and short synthesis times. The procedure works at the no-carrier-added level and gives a product of very high specific activity.     

T—2四醇和T—2五醇的制备

目的：进一步探讨Ｔ－２毒素与其结构效应的关系，制备Ｔ－２毒素的两个衍生物。Ｔ－２和Ｔ－２五醇。     

Thermodynamics of the state of a multicomponent CO2-CO-H2O-H2-N2 gas mixture in an electrolyzer with a solid electrolyte

The thermodynamic state of the gas mixture CO2 = CO = H2O = H2 = N2 in the cathode space of the electrolyzer containing a solid electrolyte is investigated. Calculation of the thermodynamic state makes it possible to determine the theoretical voltage of decomposition and concentration of individual components of this mixture at the outlet of the electrolyzer or each electrolytic cell as applied to various modes of operation. Knowledge of these parameters is important to build a technological scheme of a gas mixture regeneration system. Equations of four independent reactions are used to describe thermodynamic equilibrium reactions are used to describe thermodynamic equilibrium of the gas mixture. Particular cases that occur, when one, two or more conditions of the technological process are not satisfied, are considered.     

Identification of the two cis-syn [2 + 2] cycloadducts resulting from the photoreaction of 3-carbethoxypsoralen with 2'-deoxycytidine and 2'-deoxyuridine

Near-ultraviolet photolysis of 2'-deoxycytidine (dCyd) and 3-carbethoxypsoralen (3-CPs) in the dry state was found to generate two main stable photoadducts which were separated by thin-layer and high-performance liquid chromatography. Fast atom bombardment and plasma desorption mass spectrometry analyses suggested that the bound molecule to 3-CPs is dCyd. These two compounds were found to produce the corresponding 2'-deoxyuridine (dUrd) derivatives through a deamination process when left in aqueous solutions with a lifetime close to 24 h at 20 degrees C. The chemical structure of the deaminated photoadducts was confirmed by photochemical synthesis using dUrd as the substrate. UV and fluorescent measurements indicated that the furan moiety of 3-CPs is involved in the photobinding reaction. The cyclobutane type structure of the modified dUrd derivatives was established on the basis of its photoreversibility and detailed 1H NMR analysis. The cis-syn stereoconfiguration of the two photocycloadducts was inferred from coupling constant considerations and on the basis of the complete assignment of the cyclobutyl protons, requiring the synthesis of deuterated nucleosides at pyrimidine carbon C(6). Further confirmation of the diastereoisomeric relationship between the two cis-syn dUrd <54' 65'> 3-CPs was provided by circular dichroism measurements.     

New high-yield synthesis of 18F-labelled 2-deoxy-2-fluoro-D-glucose

A new high-yield synthesis for the production of 18F-2-FDG has been developed by reacting 18F-labelled acetyl hypofluorite, prepared by in situ reaction of 18F-molecular fluorine with sodium acetate in glacial acetic acid, and tri-acetyl-D-glucal at room temperature. Molecular fluorine labelled with 18F was produced by a 20Ne(d, alpha) 18F reaction. 1,3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-D-glucopyranose is extracted with methylene chloride, evaporated to dryness and hydrolyzed, yielding 98% radiochemically pure 18F-2-FDG. Overall radiochemical yield is about 24 +/- 3%. The specific activity of the final product at the end of synthesis is about 25.38 GBq/mmol (685 mCi/mmol). The synthesis time is approximately 60 min. The synthesis proves that small medical cyclotrons are able to produce 18F-molecular fluorine at the levels needed for the synthesis of 18F-2-FDG used in functional imaging with positron emission tomography.     

Sources of hepatic glucose production by 2H2O ingestion and Bayesian analysis of 2H glucuronide enrichment

The contribution of gluconeogenesis to hepatic glucose production (GP) was quantified after ²H₂O ingestion by Bayesian analysis of the position 2 and 5 ²H‐NMR signals (H2 and H5) of monoacetone glucose (MAG) derived from urinary acetaminophen glucuronide. Six controls and 10 kidney transplant (KTx) patients with cyclosporine A (CsA) immunosuppressant therapy were studied. Seven KTx patients were lean and euglycemic (BMI = 24.3 ± 1.0 kg/m²; fasting glucose = 4.7 ± 0.1 mM) while three were obese and hyperglycemic (BMI = 30.5 ± 0.7 kg/m²; fasting glucose = 7.1 ± 0.5 mM). For the 16 spectra analyzed, the mean coefficient of variation for the gluconeogenesis contribution was 10% ± 5%. This uncertainty was associated with a mean signal‐to‐noise ratio (SNR) of 79:1 and 45:1 for the MAG H2 and H5 signals, respectively. For control subjects, gluconeogenesis contributed 54% ± 7% of GP as determined by the mean and standard deviation (SD) of individual Bayesian analyses. For the lean/normoglycemic KTx subjects, the gluconeogenic contribution to GP was 62% ± 7% (P = 0.06 vs. controls), while hyperglycemic/obese KTx patients had a gluconeogenic contribution of 68% ± 3% (P < 0.005 vs. controls). These data suggest that in KTx patients, an increased gluconeogenic contribution to GP is strongly associated with obesity and hyperglycemia. Magn Reson Med 60:517–523, 2008. © 2008 Wiley‐Liss, Inc.     

2-CAT: 2-concentric arc technique

We used conventional three-dimensional (3D) planning software and blocking to design a radiation dose distribution in the shape of a hollow sphere. The technique, known as 2-CAT, employs arcing beams with blocking of the center of the field, along with partial transmission blocking of the poles of the portal. The central core can subsequently be irradiated to deliver a relatively even dose to the entire volume. The technique differs from conventional planning in that the central portion of the tumor, while surrounded, is not included in the initial treatment volume. The clinical usefulness of the method will require future testing, as the technique has not yet been clinically proven..     

Compton profiles and electronic structure of HgBr2 and HgI2

In this paper, we present the first-ever experimental Compton line shapes of HgBr₂ and HgI₂ using ¹³⁷Cs Compton spectrometer. To compare our experimental momentum densities, we have computed the Compton profiles using Hartree–Fock and density functional theory within linear combination of atomic orbitals. We have also computed the energy bands and density of states using the linear combination of atomic orbitals and full potential linearized augmented plane wave method. On the basis of equal-valence-electron-density profiles, it is seen that HgI₂ is more covalent than HgBr₂ which is in agreement with the valence charge densities. The experimental isotropic profiles are found to be relatively in better agreement with the Hartree–Fock data. We have also discussed the photoluminescence and detection properties of both the halides.     

兔VX2肝癌模型的MMP-2的表达

目的 探讨在兔VX2肝癌模型上的金属蛋白酶-2(MMP-2)的表达特征及其意义.方法 建立30只新西兰大白兔VX2肝癌模型.成瘤后第2.5周处死动物,取得癌组织及癌旁组织30例,正常肝组织20例,进行MMP-2的免疫组化SP染色.根据MMP-2 免疫组化染色的阳性结果进行阳性细胞计数,同时根据细胞阳性染色的数目确定阳性、阴性.结果 癌组织内MMP-2表达呈阳性者26例,阳性率为86.7%(26/30),癌旁组织表达阳性16例,阳性率为53.3%(16/30),正常兔肝组织表达为阴性.癌组织内表达部位主要在癌细胞胞浆和间质组织内,癌栓内、胆管上皮、血管内皮均有部分表达.结论 兔VX2肝癌的MMP-2表达特征与人类肝癌(HCC)的表达相似,在此模型上从MMP-2角度进行肝癌临床相关研究具有一定可行性.